Estimation of bile pigments in serum

ESTIMATION OF BILE PIGMENTS IN SERUM

VAN DEN BERGH METHOD:

PRINCIPLE:

serum bilirubin is converted to the purple-colored compound azobilirubin when reacted with diazotized sulphanilic acid (Ehrlick's reagent).

REAGENTS REQUIRED:

1. methanol (AR grade)
2.HCL(1.5%)
3.solutionA: dilute 15ml conc.HCL to 1litre with dist.water
4.solutionB: Dissolve 0.5g sodium nitrate in water and make up the volume to 100ml
5.Diazoreagent(freshly prepared): to 10ml of solutionA, add 0.3ml of solutionB
6.standard solution of bilirubin :(0.1mg/ml) in methanol.

PROCEDURE:

(A). PREPARATION OF UNKNOWN SAMPLE:

1. in a tube labeled as “u”, take 0.2ml of serum.
2. to it add van den Bergh mixture (diazo reagent) 0.5ml.
3. add 0.5ml of distilled water to the above mixture
         4. add 2.5ml of methanol
5. mix well and allow to keep for 30min
6. measure the absorbance at 540nm using a spectrophotometer.
NOTE: it is “Eu”

(B). PREPARATION OF CONTROL SAMPLE:

1. in a tube labeled as “e” take 0.2ml of serum
2. add 0.5ml of Van dem bergh solution A
3. add 1.8ml of distilled water
4. add 2.5ml of methanol
5. mix well and keep for 30 min
6. measure the absorbance at 540nm.
NOTE: it is “Ec”

C.PREPARATION OF STANDARD SAMPLE:

1. in a tube labeled as “S” take standard bilirubin solution 0.2ml
2. add van den Bergh mixture 0.5ml
3. add 4-3 ml of methanol
4. mix well and keep for 30min
5. measure the absorbance at 540nm
NOTE: it is “Es”

(D)PREPARATION OF SAMPLE FOR CONJUGATED BILIRUBIN:

in a test tube labeled as “conj” take 0.2ml of serum
add van den Bergh mixture 0.5ml
add 4.3ml of distilled water
measure the absorbance at 540nm
NOTE: it is “E conj”

       total bilirubin (mg/100ml) = Eu-Ec/Es X 5
      
         conjugated bilirubin = Econj – Ec/ Es X 5

CLINICAL SIGNIFICANCE:

normal range of serum total bilirubin is 0.0 to 1.5 mg/100ml. direct bilirubin is water-soluble conjugated bilirubin free bilirubin or water-insoluble bilirubin  hyperbilirubinemia is a characteristic of jaundice  jaundice can be classified into three different types:
1. hemolytic or preoptic jaundice (unconjugated bilirubin content is more)
2. hepatic or parenchymatous jaundice (mixture of conj and un-conj bilirubin are seen)
3. obstructive or post hepatic jaundice (conj bilirubin content is more)
total bilirubin in serum = 0 -1.5mg/100ml.
Un-conjugated = 0 – 1.25mg/100ml.
Conjugated = 0 – 0.25mg/100ml.